The Amperometric Cholesterol Sensors

Published Date: 02 Nov 2017

T Kappers, BSc PJ Porte, BSc

Abstract

This review paper gives an overview of amperometric sensors for cholesterol developed during

the last five years. Cholesterol is a chemical found in fat, blood and cells and can cause

heart attacks when levels are too high. Most sensors are based on the same reactions which is

Cholesterol + O2

ChOx ô€€€ô€€€ô€€€ô€€€! Cholest ô€€€ 4 ô€€€ en ô€€€ 3 ô€€€ one + H2O2. Several sensors are viewed and

the important working methods and features are described. This paper shows there are several

different methods for the detection of cholesterol, all with their own features.

Introduction

Cholesterol is a chemical substance found in fat,

blood, and cells in the human body and higher

animals. It is essential for human’s life since

it can build and maintain membranes of cell.

Unfortunately, high cholesterol levels are related

to cardiovascular disease such as myocardial infarction

and stroke.2 Its level in blood is an

important parameter in the diagnosis and prevention

of disease. Ideally, the total cholesterol

concentration in a healthy person’s blood should

be less than 200 mg/dL (<5.17 mM). The borderline

high is defined as 200-239 mg/dL (5.17-

6.18 mM), and the high value is defined as above

240 mg/dL (>6.21mM).8 Most cholesterol sensors

are based on the enzymatic reaction in the

use of cholesteroloxidase (ChOx) which can be

described as in equation 1 and can be seen in

figure 1.

Cholesterol + O2

ChOx ô€€€ô€€€ô€€€ô€€€!

Cholest ô€€€ 4 ô€€€ en ô€€€ 3 ô€€€ one + H2O2

(1)

Because of the increasing amount of people with

high levels of cholesterol, the cholesterol sensor

became more important. The consequence is the

development of new cholesterol sensors. Mostly

the developed cholesterol sensors are amperometric

biosensors. The amperometric biosensors

are chosen because of their good selectivity,

rapid response and low cost.8 This paper

review describes the developed amperomet-

Figure 1: Reaction of cholesterol

ric cholesterol biosensors of the last five years,

mostly based on the reaction described above.

Amperometry

Amperometry is a technique that uses electric

current to determine a concentration of a specific

ion. A reduction current, which occurs by

the flow of electrons from the electrode to the

solution, causes a reduction reaction. The reduction

reaction is part of the redox reaction

which occurs in the solution. This reaction will

cause a decrease of the electrode potential. The

electrons needed for this reaction are from a reaction

at the reference electrode. Because of this

reaction less ’oxidation’ ions are present around

the working electrode. This causes a gradient

in the ion concentration, which causes a diffusion

of the specific ions. There will arise a flux

1

to the electrode, which is related to the reducing

current. The original concentration of the

ions can be determined through the reduction

current. The reduction current is dependent on

the flux and the flux is dependent on the concentration

gradient as can be seen in equation 2.

ired = nFADox

_Cox(x)

_x

(2)

With a few assumptions this equation can be

simplified. First of all, the slope of the gradient

is linear. Secondly, the layer where the diffusion

takes place has a fixed thickness. Thirdly the

Cox(x = 0) has to be zero, for that a good potential

has to be chosen. The result of equation 2

and the assumptions above is shown in equation

3.

ired;limiting

= nFADox

_(Cox(bulk) ô€€€ Cox(x = 0))

_x

=

nFAD

_x

Cox(bulk)

(3)

With equation 3 it is possible to determine the

concentration of ions.

Sensors

For this review paper there is looked at various

sensors for the detection of cholesterol. These

sensors are described briefly and the important

features are summarized in table 1. RSD is

the sensor to sensor reproducibility relative

standard deviation.

Abdelwahab et al. used the conducting

polymer, poly3,4-diamine-2,2,5,2-thertiphene

(PDATT) as a matrix for the immobilization

of biomolecules and Gluthatione (GSH) as a

matrix for the capture of gold nanoparticles

(AuNPs) and biomolecules (ChOx).1 In this

study the process of immobilized ChOx on the

AuNPs-GSH/PDATT electrode was studied to

develop a cholesterol sensor. The maximum

current response was observed at a potential

of -0.3 Volt with an optimum pH of 7.0 and

an optimum temperature of 25°C. The performance

of the cholesterol sensor was tested under

optimum conditions and showed a sensitivity of

0.054 _A_Mô€€€1, as can be seen in figure 2, with

a linear range from 10-30 _M and a detection

limit of 0.3 _M The detection time was 0.5

s=_M with a RSD of 4.7%.

Figure 2: Amperometric response using

the ChOx/MP/AuNPs-GSH/PDATT electrode.

The inset shows thecorresponding calibration

plot of cholesterol.1

Most cholesterol sensors use cholesterol oxidase

(COD) as the enzyme to catalytically oxidize

cholesterol to 4-cholesten-3-one and hydrogen

peroxide. Disadvantages of this use are

physical and chemical influences like temperature

and pH effects. Chiang et al. detect cholesterol

based on indirect electrochemical oxidation

with bromine which acts as electron mediators

or electrocatalysts.2 A three-electrode

system was used, with a platinum plate as working

electrode, a Ag/AgCl/saturated KCl electrode

as reference electrode and a platinum plate

as counter electrode. It takes 60 seconds to

reach an equilibrium and an equilibrium potential

of 0.5 V was reached. The cholesterol

is oxidized in a electrolytic solution consisting

of NaClO4, KBr and N-N dimethyl-formamide

(DMF) in which NaClO4 acts as a supporting

electrolyte to improve the conductance. KBr

serves as a source of the bromine and DMF is

for dissolving cholesterol. Amperometric detection

was chosen at an applied potential of 1.8

2

V (vs. Ag/AgCl/saturated KCl). With this

cholesterol sensor there was found a sensitivity

of 31.08 _Ammolô€€€1cmô€€€2, a linear range of 30-

200 _M, a detection limit of 3.2 _M and a response

time of 45 seconds. The sensitivity of

this cholesterol sensor showed in figure 3.

Figure 3: Calibration curve for the amperometric

detection of cholesterol using an indirect electrochemical

oxidation method.2

The cholesterol sensor of Fang et al. has a

carbon electrode base for the working, reference

and auxiliary electrodes.3 More ingredients

should be added to the working electrode,

like cholesterol esterase, cholesterol dehydrogenase

and the coenzyme nicotinamide adenine

dinucleotide (NAD). The reactions that take

place can be seen in figure 4. This sensor is

disposable.

A buffer of 50 mM phosphate with a pH of

7.0 is used to get a required pH for a sensor

response that is as high as possible. The sensor

is tested for different temperatures. It showed

that there is only a small different in sensor

response between a temperature range from 15

to 42°C which shows this sensor is temperature

independent.

The sensor has a linearity range of 50 - 500

mg/dl and a detection limit of 50 mg/dl. The

sensitivity is equal to 0.02344 _A=(mg=dl).

This is showed in figure 5. The sensor has

a stability of 100 days. The sensor tests the

cholesterol through a simple test strip. It has

a few advantages because of its simplicity like

the low costs and the reproducibility of the

fabrication of the strips. Because it is disposable

it can easily be used in every household.

Figure 4: Schematic depiction of total cholesterol

biosensor, where CHD, cholesterol dehydrogenase;

CE, cholesterol esterase; Chol,

cholesterol; ChE, cholesterol ester; PDo is the

oxidation state of PD and PDr is the reduction

state.3

The amperometric cholesterol sensor of

Huang et al. is a non-enzyme sensor based on

the host-guest effect of Beta-cyclodextrin(_-

CD) for cholesterol.4 This host-guest effect is

used to extract the cholesterol out of a specific

solution. An Au electrode is used as base for the

working electrode. The total working electrode

was named as MB/_-CD/AuNPs/Au. Methylene

blue(MB) and Beta-cyclodextrin are added

to the electrode. The sensor uses a saturated

calomel electrode as reference electrode and

platinum wire as auxiliary electrode.

As shown in figure 6 the cholesterol replaced

the MB which was in the _-CD. And because

of this change the peak current decreases. This

means that the MB molecules diffuse into the

solution, which results in a reduction of the

redox reaction.

The disadvantages of an enzymatic sensor, like

that it is required to retain the catalytic activity

of the enzyme, will be overcome by the use of a

3

Figure 5: the calibration curves of the total

cholesterol biosensors at sampling time 38 s with

linear regressions. Five readings were taken at

each measurement.3

nonenzymatic sensor.

The highest sensor response was reached with a

pH of 7. That is why 20 mM phosphate buffered

saline (PBS) with a pH of 7.0 was chosen as the

supporting electrolyte.

When this sensor is placed in a cholesterol solution

the MB will be replaced by the cholesterol.

Because the MB is in the solution there will be

a reduction of the redox signal.

The sensor showed a linearity from 2:0 _ 10ô€€€8

to 5:0 _ 10ô€€€5 M and has a minimum detection

limit of 7 _ 10ô€€€9 M. The recovery of the determination

of the sensor has been measured to

know if the sensor could be used for real sample

determination. The recovery has a range of

96.5 to 103.6% for three serum samples, this

shows that this technique can be used for the

detection of cholesterol.

Figure 6: Schematic presentation of the fabricated

cholesterol amperometric biosensor.4

The amperometric cholesterol sensor of Li

et al. uses porous tubular silver nanoparticles

to measure cholesterol.5 This will be used for

the working electrode with a glassy carbon

electrode. The reference electrode is a saturated

calomel electrode and the counter electrode is

platinum wire. It is a nonenzymatic sensor.

An enzymatic sensor has a few disadvantages

like that the activity of an enzyme decreases

because of the use and the sensor response is

dependent of the temperature and pH. That

is why also a nonenzymatic sensor is made to

overcome these disadvantages of an enzymatic

sensor. And the porous Pt nanostructure should

be working well for this electrode.

The response can change through changes

in pH, the potential and the amount of Ag

nanoparticles on the working electrode. At a

pH of 13 the current was at his maximum. The

optimal potential is +0.35V. And the amount

of nanoparticles should be 1.5_L.

The sensor has a linear range from 2:8_10ô€€€4M to

3:3_10ô€€€2M and a detection limit of 1:8_10ô€€€4M.

This linearity is shown in figure 7. The sensor

has a good stability, after 50 measurements the

sensor response was still 95.3%. Its response

time was 20 seconds.

Figure 7: Amperometric responses of 1D porous

tubular Ag modified GCE at 0.35V upon successive

additions of 5.0_L 0.28M cholesterol to

5.0mL 0.1MpH 13 NaOH. Inset: plot of catalytic

current vs cholesterol concentration (RSD:

4.51%).5

Ohnuki et al. developed an amperometric

sensor based on hybrid organic-inorganic

Langmuir-Blodgett films.6 The integration of

organic and inorganic materials is promising for

development of new sensors and may produce

4

new functions. The Langmuir-Blodgett method

used for the development of this sensor provides

in ultra-thin multi-layer films in which thickness

can be controlled for several purposes in sensors.

In new sensors for detection glucose the

Langmuir-Blodgett is already used, Ohnuki et

al. shows that with the same technique a biosensor

for the detection of cholesterol can be made.

They developed a hybrid Langmuir-Blodgett

film of positively charged octadecyltrimethyhlammonium

(ODTA), which will immobilize the

negatively charged cholesterol oxidase (ChOx),

and nano-sized Prussian blue (PB) clusters.

This results in a hybrid Langmuir-Blodgett

film of ODTA/PB/ChOx, which works as a

cholesterol sensor. In the first step, the ChOx

catalyzes the cholesterol oxidation reaction.

In the second step, PB clusters catalyze the

reductive reaction of H2O2 at a small working

potential with a redox cycle between PB and its

reduced form of Prussian white. This induces

an electrical current flow proportional to cholesterol

concentration which can be measured with

an amperometric measurement. The cholesterol

biosensor was tested by measurement of electric

current flow, operating at 0.0 V (vs Ag/AgCl in

a buffer solution of pH 7.0). The reaction time

was found to be 20 seconds with a stable linear

relationship within the range 0.2-1.2 mmol/L.

The sensor sensitivity turned out to be 1.6

_Ammolô€€€1cmô€€€2 for a Langmuir-Blodgett film

of six layers as can be seen in figure 8.

The amperometric cholesterol sensor of Pundir

et al. is based on an epoxy resin membrane.7

The total working electrode is made of an epoxy

resin membrane with immobilized cholesterol

oxidase that was applied on a Pt electrode. The

reference electrode is an Ag/AgCl electrode and

the auxiliary electrode uses an Ag wire. There

is chosen for an epoxy resin membrane because

it should have a high affinity for enzyme, high

temperature stability, chemical resistance and

low cost.

The potential which should be used is +0.5V,

because of the highest sensor current for this

potential. The optimal pH for this sensor is 7.0

and the optimal temperature is 45°C.

The cholesterol is measured out of the serum of

Figure 8: Response current density versus

cholesterol concentration for ODTA/PB/ChOx

LB films (six layers). For the vertical axis,

changes in response current density observed at

the each cholesterol injection are plotted.6

the blood. The cholesterol sensor has a linearity

of 1-8 mM. The detection limit lies also at

1.0 mM. And the sensor has a sensitivity of

0.63_A=mM and a response time of 25 seconds.

The linearity is shown in figure 9. The stability

drops for 50% over 6 months. The coefficients

of variation were 1.59% and 1.9% and these

low values say that the method is accurate,

reproducible and reliable.

The major problem for amperometric detection

is the overestimation of the response

current due to inferences such as carbonic

acid. This problem can be overcome with

several techniques as combination of enzymes or

devising techniques to reduce the interference.

Safavi et al. use the properties of gold-platinum

(AuPt) alloy nanoparticles for fabrication of a

cholesterol sensor.8 AuPt has excellent catalysis

and resistance to deactivation due to high

synergistic action between gold and platinum.

They developed a sensor by electrodeposition of

AuPt on a glassy carbon electrode with chitosan

and ionic liquids. Chitosan is used for the excellent

film forming capability, biocompatibility,

nontoxity, good waterpermeability and high mechanical

strength. Ionic liquids are used because

5

Figure 9: Effect of cholesterol concentration on

cholesterol biosensor response based on epoxy

resin membrane bound cholesterol oxidase (inset):

Lineweaver-Burk plot of 1/I vs 1/ cholesterol

of epoxy resin membrane cholesterol oxidase.

7

of their wide electrochemical window, high ionic

conductivity and good thermal stability. ChOx

was immobilized on the surface of the electrode

by cross-linking ChOx and chitosan which resulted

in a ChOx/AuPt-Ch-IL/GCE biosensor.

The amperometric response was tested at -0.1

V and a pH of 7.0, which proved to be the best

after multiple tests. The biosensor exhibited

two linear ranges for 0.05-6.2 mM and 6.2-11.2

mM, with a sensitivity of 90.7 _Ammolô€€€1cmô€€€2

and a detection limit of 10 _M which can be

seen in figure 10. The response time of the

sensor was less than 7 seconds with a RSD of

4.2%. long-term stability was also tested with

90% of its original response after 30 days of

storage.

The amperometric cholesterol sensor of

Wisitsoraat et al. uses a functionalized carbon

nanotube(CNT) electrode as working electrode.

9 The reference electrode is made of Ag

and the auxiliary electrode of Pt. The electrodes

are placed in a flow injection microfluidic chip

based on polydimethylsiloxane/glass. The sen-

Figure 10: Calibration curve of ChOx/AuPt-

Ch-IL/GCE with different concentrations of

cholesterol.8

sor uses a microfluidic system. Its advantages

are a low sample consumption, high sample

throughput and high total analysis capability.

The CNT electrode is used because earlier

studies showed that is has a high sensitivity,

low detection potential and fast response.

The injection volume, the pH of the buffer,

the enzyme concentration and the ambient

temperature can influence the sensor response.

The optimal pH range will be between 7.0-7.5.

The best temperature range is between 20-30

°C. In both of these ranges the sensor response

is almost horizontal. The maximum enzyme

concentration is set on 50 U/ml , because

up to this point the increase is linear. The

amperometric cholesterol detection is used with

the ranges determined above.

The cholesterol sensor has a linearity between

50-400 mg/dl. The sensitivity is equal to 0.0512

nA/(mg/dl). This is shown in figure 11. The

corresponding detection limit of this sensor is

10 mg/dl. It has a throughput of around 60

samples per hour.

The amperometric cholesterol sensor of Yang

et al. is made of platinum nanoparticles.10 For

the working electrode this was added to a carbon

nanotube thin film. An Ag/AgCl plate in saturated

KCl solution was used as a reference electrode

and a platinum plate was used as counter

6

Figure 11: Calibration curve as a function of

cholesterol concentration.9

electrode. It is a nonenzymatic sensor. The advantages

of a nonenzymatic sensor are their stability,

simple fabrication and low costs and their

reproducibility. Carbon nanotubes have a high

electrical conductivity. The platinum nanoparticles

have the advantage it has a long term cyclic

stability and a Pt NP/CNT electrode can have

a wide linearity with a low detection limit and

high sensitivity. The sensor was performed in 50

mM phosphate buffered saline (PBS) with a pH

of 7.0.

The highest sensor response was reached with a

24 bilayer of the CNT film. Also the highest sensitivity

is found at a 24 bilayer. The sensor has

a linearity from 0.005 to 10 mM with a minimal

detection limit of 0.0028 mM. It has a sensitivity

of 8.7 muA/(mM cm2). This linearity is shown

in figure 12. The sensor has a good stability.

No obvious changes were seen in a period of 1

month.

Conclusion

Amperometric cholesterol sensors are made in

several different ways with all different working

principles and features. Which cholesterol sensor

is best differs on the purpose of your use and

the requirements someone is looking for. Cholesterol

concentrations in humans are defined as

high when the cholesterol sensor is above 240

mg/dL (>6.21mM). For the detection of choles-

Figure 12: Calibration plot for current of Pt

NP/(CNT)24 bilayer electrode as a function of

cholesterol concentration. Applied potential is

0.7 V.10

terol in human there is need of a cholesterol

sensor at least linear in the range of interest.

Different sensors in this review paper are not

linear within this range, but can be useful for

the determination of cholesterol concentration in

food. The concentration of cholesterol in food

might be lower than in humans and might require

a better sensitivity for instance. The decision

which cholesterol sensor one should use is

dependent on the several features which can be

seen in table 1. A decision could be made on

the importance of the features for the purpose

of the cholesterol measurement.

For this review paper there is looked at amperometric

sensors, which are the most used sensors

for the detection of cholesterol. Amperiometric

cholesterol sensors are the biggest group

of cholesterol sensors developed in the last five

year. For further research it might be of interest

to review different type of cholesterol sensors

and compare these different types.